Leishmaniasis is a vector-borne zoonotic disease endemic to the Middle East, Asia, Africa and Central America. C3HeB/FeJ (C3H) mice infected with Leishmania major develop a CD4+ TH1 response and resolve infection, while infection with Leishmania amazonensis stimulates a poor T cell response, resulting in chronic disease. When C3H mice are co-infected with both species of Leishmania we observe a healing phenotype similar to infection with L. major alone. In contrast, co-infected C57BL/6 (B6) mice have a non-healing phenotype similar to infection with L. amazonensis. We have developed an in vitro assay and have found that CD4+ T cells and B cells from the draining lymph node L. major-infected C3H mice placed in this transwell assay are sufficient and necessary for macrophage activation and killing of L. amazonensis. When [unreadable] B6 mouse-derived total lymph node cells or purified CD4+ T cells and B cells from a B6 mouse infected with L. major are co-cultured in vitro with L. amazonensis infected macrophages parasite killing is not observed. A series of cell transfer studies have demonstrated that B cells from the B6 mice do not promote killing of intracellular L. amazonensis. These findings from our in vitro parasite killing assay and in vivo transfer experiments indicate that B cells play a prominent role in the cell mediated immune response against L. amazonensis and that B cells from B6 mice are unable to promote macrophage parasite killing compared to cells from CSH mice. We have generated two hypotheses concerning the difference between C3H and B6 B cells. First, we hypothesize B cells from C3H mice produce pro-inflammatory cytokines that positively influence CD4+ T cells to activate co-infected macrophages, while B cells from B6 mice produce anti-inflammatory cytokines that negatively influence CD4+ T cells to activate infected macrophages. Our second hypothesis is the production of the novel antibody isotype lgG2c by B6 mice may inadequately stimulate infected macrophages via the Fc gamma receptors. [unreadable] [unreadable] The work in this proposal will 1) establish if B cells from C57BL/6 mice inadequately stimulate a productive Th1 immune response during co-infection by phenotypically characterizing B cells; and 2) determine if lgG2c production by B6 B cells alters macrophage activation. Results of this work will emphasize the importance of B cells in Leishmania infection and all will provide a foundation for development of treatment and vaccine options of cutaneous leishmaniasis in animals and humans. These findings will also advance our understanding of genetic predispositions to leishmaniasis. [unreadable] [unreadable] [unreadable]